Prize-Winning PhD

“It just wasn’t a recognized scientific problem that a lot of people were working on,” notes Harvard’s Dan Finley. “Few labs were interested.”

But Ciechanover and his advisor, Avram Hershko, were. They worked on the problem at the Technion-Israel Institute of Technology in Haifa and during summer sabbaticals in the laboratory of Irwin “Ernie” Rose at the Fox Chase Cancer Center in Philadelphia. In just 5 short years, they sorted out the major players in the pathway—and began to unravel the effects that ubiquitin plays on protein degradation. “This was his PhD thesis,” notes Goldberg.

That achievement opened up a new understanding of a fundamental process that “influences every major biological function in the cell: the cell cycle, apoptosis, intracellular trafficking, signal transduction—basically everything,” says Maria Masucci of the Karolinska Institute in Stockholm. “And when things go wrong, it can lead to cancer, autoimmunity, neurodegeneration, you name it.”

“Because Aaron was a student when the work was done, I think he probably gets less credit than Hershko and Rose did,” says Keith Wilkinson of Emory University, who was a postdoc in Rose’s lab at the time. “But he was the one doing the hands-on work.” And his name is on every one of the key papers. “It’s hard to distinguish between the contributions of the student and the advisor,” adds Goldberg. “But it’s also hard to imagine either of them achieving this alone.”

Ciechanover came to science from a medical background. He received his MD from the Hebrew University in Jerusalem in 1972, then served as a combat physician in the Israeli army. “Life takes you wherever,” he says of the switch. “I started with medicine, and had some thoughts about surgery, but I fell in love with science.”

Though the intensity of the operating room can be exciting, the adrenaline rush—and the involvement—tend to be short-lived. “You operate on one patient, then you go to the next,” says Ciechanover. “But science is forever. It’s a long-standing love.”

That love of science blossomed once Ciechanover had completed his compulsory military service. When he signed up for grad school in 1976, Hershko was already working on the mechanism by which cells dismantle their proteins. The investigators were following up on Goldberg’s discovery that proteins could be degraded in a regulated manner—requiring an input of energy—in the cell cytoplasm. Before then, researchers thought that protein demolition was confined to lysosomes: organelles that are essentially little sacks of enzymes that digest nearly everything in sight, with no added energy needed. But such indiscriminate disassembly could not explain why some proteins have short half-lives while others hang around forever, or why starvation and other stresses can affect proteins’ stability. “To achieve such exquisite specificity, there had to be more subtle biochemistry going on,” says Goldberg.

Inside the cell, the biochemistry might have been subtle. But in the lab, it was anything but. “It was real bucket biochemistry,” says Wilkinson. “We didn’t have the analytical techniques, the sensitivity, we have today. In those days you needed maybe 1000-fold more material to carry out the sort of analysis that can be done today in a heartbeat.”

And Ciechanover was a believer in doing things big. “We had this quantity we called an ‘Aaron mole’—a mole being a large amount of something, and an Aaron mole being an even larger amount,” says Wilkinson. “He would make things on very large scales, and he’d spend the time and energy needed to tackle a problem”—even if it meant the shirt off his back. “In those days, the labs in Fox Chase weren’t air conditioned,” says Wilkinson. “And the summer heat in Philadelphia can be significant. So Aaron took to coming in, taking off his shirt, and basically working bare chested in his lab coat.”

The researchers were working with reticulocytes, immature red blood cells chosen for these studies because they lack lysosomes. Thus, any protease activity the investigators might find there would have to occur outside the lysosome. They broke open the cells and first separated their contents into two fractions: one that contained the negatively charged proteins that got stuck to an anion exchange column, and another that included the remaining proteins in the cell. And they found that neither fraction was capable of digesting a “test” protein on its own. But put back together, they performed the reaction just fine. That meant that each fraction contained a component—or components—necessary for carrying out protein degradation.

To isolate those components, the team started with the flow-through fraction, which was dark red because it contained the cells’ hemoglobin. By boiling the sample, Ciechanover eliminated the hemoglobin, leaving behind a small, heat-stable protein capable of driving protein degradation. In another experiment, the investigators found that this protein becomes physically linked to other proteins. Then one evening over beers, Wilkinson told Mike Urban, a postdoc from a neighboring lab, about this small protein that becomes attached to other proteins. “And he told us about ubiquitin,” Wilkinson says—a small stable protein that was known to covalently link to a protein called histone H2A.

The two proteins—ubiquitin and the mystery protein from fraction 1—were, indeed, one and the same. With that protein in hand—and the knowledge of its identity—Ciechanover, Hershko, and their colleagues were then able to isolate the other components of the system: a trio of enzymes that activate ubiquitin and ultimately tack it on to proteins destined for degradation. “It was like peeling an onion,” says Ciechanover. “We didn’t know what the next layer would be. We just worked it out step by step, using nothing more sophisticated than a fraction collector, a centrifuge, and an affinity column with ubiquitin attached. It was really simple, basic biochemistry at its best. Embarrassingly simple!”

But also spot on. “It’s really quite amazing the amount of inference that turned out to be correct, based on some pretty simple sorts of classical biochemical approaches,” says Wilkinson. And the biochemistry was soon backed up by genetics, as Alexander Varshavsky of MIT began to identify the yeast genes that encode these ubiquitin-handling enzymes. In fact, Ciechanover teamed up with Varshavsky during an action-packed postdoc in Harvey Lodish’s lab in the early 1980s. “I was his postdoctoral advisor,” says Lodish. “But he didn’t need much advice.” Working with fellow postdoc Alice Dautry, Ciechanover unraveled the mechanism by which the transferrin receptor brings iron into the cell, a study Lodish dubs “a spectacular piece of work.”

At the same time, Ciechanover continued to moonlight on the ubiquitin project. “He was a real ball of fire,” says Finley, who was a student in Varshavsky’s lab. “He loved ubiquitin with an unholy passion and he expressed that at the bench by his willingness to basically do anything. In those days, the technology was pretty primitive, so we had to dive in and do things ourselves rather than just pressing a button. We worked really hard to get everything right and never doubted that we were doing something of vast importance.”

They were studying a mutant cell type that, when subjected to elevated temperatures, failed to ubiquitinate histone H2A. And they found that the mutation fell in the ubiquitin-activating enzyme. As a result, these mutant cells also had trouble disposing of their proteins. “And it was evident that this was the pathway controlling protein degradation,” says Finley.

“Those were probably some of my best years in science,” says Ciechanover. “I could just work at the bench and follow my ideas. If someone offered to pay for my salary and my expenses, I would happily become a postdoc again.”

In 1984, Ciechanover returned to the Technion to set up his own lab, where he has continued to study how the ubiquitination system recognizes target proteins and tags them for demolition. He’s shown that ubiquitin can be degraded along with its target protein. And that ubiquitin can be attached to the N-terminal end of a protein, not just to its internal lysine residues, as had previously been assumed. “This is a concept that had been ruled out, and through Aaron’s enthusiastic work has now been ruled back in,” says Goldberg.

“He doesn’t give up,” says Shai Cohen, who works with Ciechanover at the Technion. “When he believes in something he’ll keep working on it for years, even if it makes no sense to anyone else.” In the case of N-terminal ubiquitination, “at first some people didn’t believe it, and others thought it was some esoteric anecdote,” says Michael Glickman, also at the Technion. “But now I think it’s generally considered canon—and one way for cells to destroy proteins that lack lysine residues.”

“Paradigms don’t last forever,” notes Ciechanover. “You always shatter them. So the idea is not to believe in paradigms. Just do the experiments and see what the results are.”

Ciechanover continues to follow where the experiments take him. And winning a Nobel has not made him any less busy. “He works even harder now,” says Cohen. “But I think he is less tense. When the community says your findings are a major thing, you no longer have to prove, again and again, that your work is important. Which is a kind of relief. Of course, you still have that drive to find new things.”

“You can’t get up every morning for years and celebrate,” says Ciechanover. “You have to get back to work and do something.” But being a Laureate does have its benefits. “I get to travel to interesting places and meet interesting people,” he says. And though receiving the chemistry prize was a bit of a surprise—“I’m not a chemist, I never trained in chemistry,” Ciechanover says—“we didn’t appeal the decision.”